Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease with high prevalence in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec. Disease progression is rapid through young adulthood, with most patients requiring wheelchairs by their early forties. The disease is characterized by abolished sensory nerve conduction, reduced motor nerve velocity, and a unique clinical feature of hypermyelination of retinal nerve fibers. Additional pathological features include atrophy of the upper cerebellar vermis, absence of Purkinje cells, and possibly abnormal neuronal lipid storage (Bouchard, J -P., In: Handbook of Clinical Neurology 16: Hereditary neuropathies and spinocerebellar degenerations, J.M.B.V. de Jong, Ed., pp. 451-459, Elsevier Science Publishers, Amsterdam (1991)). A developmental defect in the myelination of both retinal and peripheral nerve fibers has been proposed as the physiological basis of the disease (Bouchard, J -P., et al., Neuromuscular Disorders 8:474-479 (1998)). More than 300 patients have been identified, and the estimated carrier frequency is 1 in 22 in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) population of northeastern Quebec (3).
As described herein, the ARSACS gene, referred to herein as xe2x80x9cspastinxe2x80x9d (also known as sacsin), has been mapped to chromosome 13q11 by linkage analysis and cloned from human, mouse and hamster. The gene was identified by using fine-structure linkage disequilibrium (LD) mapping to narrow the disease interval and then performing sample-sequencing to identify candidate genes. The spastin gene has a remarkable feature in that it contains a large exon spanning at least 12,793 base pairs of genomic DNA and comprises an open-reading frame of 11,487 base pairs. As described herein the gene is highly conserved in mouse. This exon of spastin is the largest found in any vertebrate organism. The deduced protein contains three large domains with sequence similarity to each other, as well as to the protein predicted to be encoded by an open reading frame identified in Arabidopsis genomic DNA. These domains contain a subdomain with sequence similarity to heat-shock proteins, suggesting a role in chaperone-mediated protein folding. Spastin appears to be expressed in a wide variety of tissues including brain and central nervous system. Alterations in the spastin gene have been identified as described herein which correlate strongly with ARSACS, including at least two alterations which have severe effects on the encoded protein, providing strong evidence that mutations in the open reading frame of the spastin gene are responsible for ARSACS.
The present invention relates to an isolated nucleic acid molecule comprising a spastin gene or portion of said gene as described herein. In one embodiment, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15 and the complement of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15. In another embodiment the invention relates to an isolated nucleic acid molecule comprising an exon from a vertebrate gene wherein said exon is at least 1150 base pairs in length. The invention also relates to an isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15 and the complement of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15. In a preferred embodiment the genes of the invention are human genes. The invention also relates to an isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 21-66 and the complement of SEQ ID NOS: 21-66.
The present invention also includes fragments of the spastin genes described herein. For example, the invention relates to an isolated portion of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15 and the complement of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15, wherein the portion is at least about 10 nucleotides in length.
The invention also relates to nucleic acid molecules having substantial sequence identity to the specific sequences disclosed herein. In one embodiment, the invention relates to a nucleic acid molecule comprising a nucleotide sequence which is at least about 60% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15 and the complement of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15. In another embodiment, the invention relates to a nucleic acid molecule which hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15 and the complement of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14 and 15.
The nucleic acid molecules of the present invention, or portions thereof, can be used as probes to isolate and/or clone substantially similar or functionally equivalent homologues of the spastin family of genes. The polynucleotides of the present invention can also be used as probes to detect and or measure expression of the genes encoded by the present invention. The probes of the present invention can be DNA, RNA or PNA. Expression assays, such as Southern blot analysis and whole mount in situ hybridization, are well known in the art. The polynucleotides of the present invention, or portions thereof, can also be used as primers to clone homologues or family members by PCR using techniques well known in the art.
The invention further relates to nucleic acid constructs comprising the isolated nucleic acid molecules of the invention, as well as to a recombinant host cell comprising the isolated nucleic acid molecules of the invention. The invention further relates to a method for preparing a polypeptide encoded by an isolated nucleic acid molecule of the invention, comprising culturing the recombinant host cells of the invention.
Also encompassed by the present invention are isolated polypeptides encoded by nucleic acid molecules described herein. For example, the invention relates to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 8, 10, 16 and 67-69. The invention also relates to an isolated polypeptide comprising an amino acid sequence having greater than 75% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 8, 10, 16 and 67-69. The invention also provides antibodies, and antigen binding fragments thereof, to the polypeptides of the invention, particularly antibodies and antigen binding fragments thereof which specifically bind the polypeptides described herein.
The invention also provides a method for assaying the presence of a nucleic acid molecule in a sample, comprising contacting said sample with a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14, 15, 17-66, 72 and 73; the complement of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14, 15, 17-66, 72 and 73; a portion of any one of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14, 15, 17-66, 72 and 73 which is at least 10 nucleotides in length; and a portion of the complement of any one of SEQ ID NOS: 1, 3, 7, 9, 11, 12, 13, 14, 15, 17-66, 72 and 73 which is at least 10 nucleotides in length, under conditions appropriate for selective hybridization of the sequence to the nucleic acid molecule in the sample. Presence or absence of a hybridization signal indicates presence or absence, respectively, of the target nucleic acid molecule. The invention also relates to a method for assaying the presence of a polypeptide encoded by an isolated nucleic acid molecule of the invention in a sample, comprising contacting said sample with an antibody which specifically binds to the encoded polypeptide.
The invention further relates to a method of diagnosing or aiding in the diagnosis of neurodegenerative disease in an individual comprising obtaining a nucleic acid sample from the individual and determining the nucleotide present at nucleotide position 5254 of SEQ ID NO: 1, wherein the nucleotide position is numbered from the putative initiation codon, wherein presence of a thymine at said position is indicative of increased likelihood of neurodegenerative disease in the individual as compared with an appropriate control, e.g., an individual having a cytosine at said position. The invention also relates to a method of diagnosing or aiding in the diagnosis of neurodegenerative disease in an individual comprising obtaining a nucleic acid sample from the individual and determining whether there is a deletion of a thymine at nucleotide position 6594 of SEQ ID NO: 1, wherein the nucleotide position is numbered from the putative initiation codon, wherein deletion of a thymine at said position is indicative of increased likelihood of neurodegenerative disease in the individual as compared with an appropriate control, e.g., an individual who does not have a deletion at said position.
The invention also relates to a method of treating a neurodegenerative disorder associated with the presence of a thymine at nucleotide position 5254 of SEQ ID NO: 1 in an individual, wherein the nucleotide position is numbered from the putative initiation codon, comprising administering to the individual an agent selected from the group consisting of a polypeptide encoded by SEQ ID NO: 2 or an active portion thereof, a nucleic acid molecule which encodes SEQ ID NO: 2 or an active portion of SEQ ID NO: 2, and an agonist of SEQ ID NO: 2. The invention further relates to a method of treating a neurodegenerative disorder associated with a deletion at nucleotide position 6594 of SEQ ID NO: 1 in an individual, wherein the nucleotide position is numbered from the putative initiation codon, comprising administering to the individual an agent selected from the group consisting of a polypeptide encoded by SEQ ID NO: 2 or an active portion thereof, a nucleic acid molecule which encodes SEQ ID NO: 2 or an active portion of SEQ ID NO: 2, and an agonist of SEQ ID NO: 2.
The invention also encompasses a method of diagnosing or aiding in the diagnosis of neurodegenerative disease associated with the presence of a thymine at nucleotide position 5254 of SEQ ID NO: 1 in an individual, wherein the nucleotide position is numbered from the putative initiation codon, comprising obtaining a sample comprising a Spastin polypeptide from the individual and determining the size of the Spastin polypeptide, wherein if the Spastin polypeptide is significantly shorter than SEQ ID NO: 2 it is indicative of neurodegenerative disease. The invention also provides a method of diagnosing or aiding in the diagnosis of neurodegenerative disease associated with the presence of a deletion at nucleotide position 6594 of SEQ ID NO: 1 in an individual, wherein the nucleotide position is numbered from the putative initiation codon, comprising obtaining a sample comprising a Spastin polypeptide from the individual and determining the size of the Spastin polypeptide, wherein if the Spastin polypeptide is significantly shorter than SEQ ID NO: 2 it is indicative of neurodegenerative disease. In one embodiment, the Spastin polypeptide is significantly shorter than SEQ ID NO: 2 if the Spastin polypeptide comprises less than about 75% of the amino acids of SEQ ID NO: 2.
In one embodiment, the neurodegenerative disease comprises one or more symptoms selected from the group consisting of: reduced sensory nerve conduction, reduced motor nerve velocity, hypermyelination of retinal nerve fibers, atrophy of upper cerebellar vermis, absence of Purkinje cells and abnormal neuronal lipid storage. In a particular embodiment, the nucleic acid sample is obtained from a tissue selected from the group consisting of: brain tissue, CNS, lung, fetal lung, testis, lymphocytes, adipose, fibroblasts, skeletal muscle, pancreas, uterus, kidney, tonsil, embryo and isolated cells thereof. For example, brain tissue can be selected from the group consisting of cerebral cortex, granular cell layer of the cerebellum and hippocampus. In a particular embodiment, the neurodegenerative disease is an early onset neurodegenerative disease.